The embryonic vertebrate heart has a simple anterior- posterior pattern: it is divided into two major chambers, an anterior ventricle and a posterior atrium. These two chambers are morphologically distinct; furthermore, the ventricular myocardium differs from the atrial myocardium by many molecular, histological, and physiological criteria. These intrinsic differences between the ventricle and the atrium are critical for proper cardiac function, and neonatal and adult cardiac conditions are often associated with chamber formation defects. Despite the functional importance of proper cardiac patterning, its genetic regulation is not yet understood. Through studies of cardiac chamber formation in the zebrafish embryo, we are identifying the genes that specify ventricular and atrial cell fates. In this proposal, we use zebrafish mutations to determine the roles of three key players in cardiac patterning: the transcription factor Hand2, the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2 (Raldh2), and an unidentified gene (heart of darkness (hod)). First, by creating fate maps for ventricular and atrial progenitors in wild-type and hand2 mutant zebrafish embryos, we will test models regarding the cellular function of Hand2 during myocardial differentiation and ventricle formation. Second, studies of raldh2 mutant embryos, including phenotypic characterization, fate mapping, and mosaic analysis, will test the hypothesis that retinoic acid signaling regulates atrial specification. Third, similar molecular and cellular analyses of the hod mutant phenotype will test the hypothesis that hod, like hand2, regulates early steps of myocardial differentiation and ventricle formation. Finally, we will clone the hod gene and begin the analysis of the hod gene product. Altogether, these experiments will provide a foundation for understanding the genetic pathways that regulate cardiac patterning in vertebrates.